Chimeric gas vesicle and protein expression system therefor

ABSTRACT

The present disclosure features the chimeric gas vesicle (CGV) and its expression in E. coli. The chimeric gas vesicle comprises two or more gas vesicle rib proteins. The heterologous peptides, 6-AA to 56-AA long, can be inserted into one recombinant rib protein in frame. The resulting CGV carrying the heterologous peptide can be used in many applications.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of provisional application Ser. No.63/191,902, filed May 21, 2021.

FIELD OF THE INVENTION

The invention is related to chimeric gas vesicles (CGVs) and usesthereof.

BACKGROUND OF THE INVENTION

Gas vesicles are intracellular, protein-coated and hollow organellesfound in cyanobacteria, soil bacteria and halophilic archaea. They arepermeable to ambient gases by passive diffusion and provide buoyancywhich enables bacteria cells to adjust their vertical position inaqueous environments for access of oxygen and light.

Gas vesicles are cylindrical-shaped with conical-ends nanostructures of40 to 250 nm in width and 50 to 1000 nm in length. The discrete proteinnanoparticles with permeability to ambient gas provides great potentialfor bioengineering for a variety of applications. Development ofbiological imaging under resonance and expansion of biomass productioncounting on its positive increase of buoyancy has been described in avariety of prior arts. Besides, the nano-scale bio-particles aredesirable vehicles for vaccines.

Disclosed in Pat. W01990010071A1 are recombinant cells and a recombinantvector for gas vesicle protein expression in E. coli and improvement offloatation properties of the transformed cells. Bacillus thuringiensisisraelensis (Bti) is a group of bacteria which can be used as biologicalcontrol agents for larvae stages of certain dipterans. When transformedwith gas vesicle genes, the tendency of Bti to settle in water isreduced and its larvicidal activity becomes more persistent. Other hostsovercome sedimentation problems in bio-reactors with incorporation ofgas vesicle genes, which improves bio-degradation of organic wastes ormedical/agricultural products.

In Pat. W02018043716A1, Mizushima and Watanabe published a method forintroducing gas vesicle genes including gvpA and gvpC into eukaryoticcells for gas vesicle protein expression. The gvpA and gvpC genes arederived from cyanobacteria and featured with optimized codons formammalian expression. A transposon vector serves as the vehicle of gvpAand gvpC genes for introduction of genes into mammalian cells. Themethod disclosed in this prior art also provides a kit for establishinga stable clone or a transgenic animal for constant production of gasvesicles.

In US Pat. US20140234904A1, Stephen and Levi disclosed methods forharvesting photosynthetic unicellular organisms and the formation of gasvesicles in the photosynthetic unicellular organisms. Theself-assembling gas vesicle proteins form conical filaments which arecapable of blocking water molecules but allow the diffusion of gas.Overexpression of the gas vesicle increases positive buoyancy of thephotosynthetic unicellular organisms and allows for harvesting withoutthe need for centrifugation, filtration, or chemical flocculation.

Shapiro et al (Science 27 Sep. 2019: Vol. 365, Issue 6460, pp.1469-1475) described a protocol for gas vesicle isolation andfunctionalization. The nanostructures of gas vesicles are modified withmoieties for targeting and fluorescence, and using ultrasound andmagnetic resonance for imaging. It takes 3 to 8 days for preparation ofthese genetically encodable nanostructures, and the nanostructuresenable multi-modal and noninvasive biological imaging featuring highsensitivity and potential for molecular targeting.

In Pat. W02021041934A1, Jin et al disclosed an engineered proteasesensitive gas vesicle. The engineered protease sensitive gas vesiclecomprises a GvpA or GvpB protein and an engineered GvpC protein.Protease recognition sites are inserted within a central portion of theGvpC or attached to the N or C-terminus of the GvpC. The engineered gasvesicles exhibit an ultrasound response with baseline nonlinearity tocollapse. Proteases bind to the recognition sites and result in cleavagethereof. Subsequently the engineered gas vesicles collapse following thecleavage and detection of the collapses is for screening engineeredprotease sensitive gas vesicles. The method enables reduction ofcollapse pressure profile, protease degradation, and sufficient proteinyield is obtained with improvement of engineered gas vesicle expressionin host cells. Also, ultrasound applied to obtain detection and sensingpresents an enhanced nonlinear behavior.

In US Pat. U.S. Pat. No. 5,824,309, DasSarma et al disclosed a methodfor producing protein vaccines by utilizing recombinant gas vesicles.Foreign epitopes derived from pathogens are inserted into gvpA or gvpCfor antigen presenting to elicit immunogenicity of subjects uponadministration. In absence of an adjuvant, vaccination with therecombinant gas vesicles result in long-lasting immunoglobulin IgG orIgM responses. Archaeon cells Halobacterium halobium are cultured as ahost for the engineered gas vesicle preparation.

Majority of GV research was conducted in halobacteria, cyanobacteria andPriestia megaterium (previously known as Bacillus megaterium).

In cyanobacteria and halobacteria, the lipid-free, rigid proteinaceousmembrane consists exclusively of proteins: the major hydrophobic 70 to85 amino acid (AA) GvpA protein, which forms GV ribbed basic structure,and a minor constituent, hydrophilic 200- to 450-AA GvpC, located on theGV outer surface as shown in FIG. 1. GvpC is called scaffold structuralprotein as its role in strengthening the GV structure. In soil bacteriaP. megaterium, an 88-AA GvpB protein, homologous to GvpA ofcyanobacteria and halobacteria, is found necessary for the GV formationand play the role as GvpA of cyanobacteria and halobacteria. An extracopy of gvpA gene, homologous to gvpB in P. megaterium is present in the14 gyp gene operon in P. megaterium as shown in FIG. 6 and its role isnot determined. No GvpC homolog is present in P. megaterium.

Besides the major GV structural protein GvpA and minor GV scaffoldstructural protein GvpC, five accessory GV proteins: GvpJ, GvpM, GvpF,GvpG, GvpL are detected by immunoblotting in the GV of halobacteria asindicated in FIG. 1. These accessory Gyp proteins constitute a verysmall portion of total GV protein and are hypothesized to influence theGV curvature or localized to the conical tips of GV. In thecyanobacterium A. flos-aquae , GvpJ, GvpK and GvpL homologs have beenidentified, while in P. megaterium, all five accessory GV proteinhomologs have been identified.

Gas vesicle protein A (GvpA) is the major structural protein of gasvesicles and constitutes the majority (97%) of the gas vesicle wall. Thewall is 2 nm thick and consists of a single layer of GvpA. GvpA forms4.6 nm ‘ribs’ that run nearly perpendicular to the long axis of the gasvesicle. As demonstrated in FIG. 4a , GvpA has a hydrophobic a-helix,β-sheet, β-sheet and a-helix core amino acid sequence (GvpA coresequence).

GvpA protein encoded by gvpA genes from several dozens of differentmicroorganisms are sequenced and identified. The GvpA protein sequencesare highly homologous, especially in the α-helix, β-sheet, β-sheet anda-helix core sequence as indicated in FIG. 4a . The N- and C-terminalsequences of GvpA vary among different species.

Multiple identical copies of gvpA gene are found in some cyanobacteria,two copies of slightly different gvpA gene are found on two gyp operonslocated on its chromosome and plasmid separately in halobacteria (c-vacand p-vac) and on a 14 gene gyp operon in P. megaterium (gvpA and gvpB).

It has been long recognized by the research field that there is only onekind of rib protein GvpA in each gas vesicle. In Halobacteriumsalinarum, c-vac and p-vac are located on different gene clusters atchromosome or plasmid respectively. The expression of c-vac and p-vac isunder control by different mechanisms and thus the gas vesicle formedwith either GvpA from c-vac or GvpA from p-vac at one time, not both. InP. megaterium, gvpA and gvpB are on the same 14-gene gyp operon. OnlygvpB is needed to form the gas vesicle and the role of gvpA is unclear.

Attempts are made to modify GvpA by insertion of heterologous peptidesequence into the N-terminal, C-terminal, or core sequence of GvpA.However, the rigid nature of GvpA protein limits the capacity of GvpAmodification. DasSarma's group revealed that in-frame attachment of morethan 5 heterologous amino acids to the C-terminal of GvpA in H.salinarum result in its incapability of forming gas vesicles. Otherinsertion attempts at N-terminal, and core sequence of GvpA are failedas well. This limits the use of gas vesicles in various applicationssuch as vaccines, biomarkers, etc.

GvpC is a minor scaffold structural protein and found on the gas vesiclesurface in cyanobacteria and halophilic archaea. It accounts for 2.9% oftotal protein content of gas vesicles from Anabaena flos-aquae and itcan be stripped from gas vesicles by detergent or the change of saltconcentration. It is a scaffold protein which stabilizes gas vesiclesthrough protein-protein interaction with GvpA. Attachment ofheterologous peptides to GvpC can be up to 398 amino acids long in H.salinarum, which makes GvpC more applicable in bioengineering. The gasvesicles with the modification of GvpC are called gas vesiclenanoparticles (GVNPs).

Nonetheless, attachment of GvpC to gas vesicles is not stable and theratio of GvpC/GvpA or GvpC/GV in gas vesicles is difficult to maintainconstant in practice. For instance, the GVNP produced by halobacteriacould only maintain stability in a high-salt environment. In a low-saltenvironment, GVNPs tend to break down its structure and GvpC detachesfrom the nanoparticles. Instability of GVNP and disassociation of GvpCfrom GVNP undermines the reliability of GVNPs as an antigen-presentingvehicle or imaging biomarker particles for ultrasound and MRI exam.

In another aspect, the production cycle of gas vesicles in halobacteriais 3 to 4 weeks long and time-consuming, therefore the GV/GVNPproduction cost in halobacteria is way higher than using the E. coliexpression system. Also there is lack of an efficient vector/host systemfor GV expression and production in halobacteria.

Thus, a new form of genetically engineered chimeric gas vesicle (CGV) isdesired to overcome these limitations.

SUMMARY OF THE INVENTION

In the gas vesicle research field, it is widely believed that only oneprotein, GvpA, forms the rib of the gas vesicle. In other words, in agiven gas vesicle, there is only one kind of rib protein such as GvpAfrom cyanobacteria, halobacteria and soil bacteria, or its homolog GvpBfrom P. megaterium. In the present invention, a chimeric gas vesicle(CGV) involving two or more kinds of rib proteins is disclosed.

The advantages of the chimeric gas vesicles (CGVs) of the presentinvention over traditional GVs and GVNPs are numerous. For example,previous attempts of direct genetic modification of GvpA in GVs areunsuccessful due to rigidity of GvpA in the GVs, and only a limitednumber of heterologous peptides can be inserted without the destructionof the GV. The CGV disclosed in the present invention overcomes theselimitations and makes the gas vesicle more versatile for variousapplications. In halobacteria, the genetically modified GvpC of GVNPattaches to GV via protein-protein interaction, which is vulnerable tothermohaline fluctuations and makes GvpC easy to fall off from GVNP. Inthe present invention, the heterologous peptide inserted in the CGV iscovalently fused within the rib protein, which eliminates the fall-offproblem of the heterologous peptide present on the CGV. Further, theprocess for making the CGV can be accomplished within a very short timeperiod, which drastically reduces the production cycle time compared tothe time required to make GVNPs in halobacteria or GVs in cyanobacteria.The CGVs are more stable than any other GVs previously described. Inother words, the CGVs of the present invention have a higher “criticalcollapse pressure” than the traditional GVs. In some examples, the CGVsare stable at room temperature for at least one month and at 4° C. forat least six months without any degradation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon graph to illustrate native gas vesicle as previouslydiscovered.

FIG. 2 is a cartoon graph to illustrate chimeric gas vesicle (CGV) withtwo or more different kinds of gas vesicle rib proteins in the presentinvention.

FIG. 3 is a cartoon graph to illustrate CGVs with heterologous peptides.

FIG. 4a shows alignment of gas vesicle rib proteins.

FIG. 4b shows alignment of recombinant minor rib protein withheterologous peptide.

FIGS. 5 to 6 demonstrate genetic manipulation of gyp operons for CGV.

FIG. 7 demonstrates Western Dot Blot for confirmation of CGVs carryingvarious heterologous.

FIG. 8a is an electron microscopic graph of negative stained gasvesicles and the scale bar is equivalent to 200 nm.

FIG. 8b is an electron microscopic graph of negative stained chimericgas vesicles and the scale bar is equivalent to 200 nm.

FIG. 9 is a flowchart illustrating a method for producing a chimeric gasvesicle (CGV).

FIG. 10 is a flowchart illustrating a method for eliciting immuneresponse by administrating CGV-Covid-19 to a subject in need.

DETAILED DESCRIPTION OF THE INVENTION

In the gas vesicle research field, as shown in FIG. 1, it is widelybelieved that only one protein, GvpA, forms the rib of the gas vesicle.In other words, in a given gas vesicle, there is only one kind of ribprotein GvpA, such as from cyanobacteria, halobacteria and soilbacteria, or its homolog GvpB from P. megaterium, to be found.

In one aspect, the present invention provides a chimeric gas vesiclecomprising at least two Gyp rib proteins, wherein the rib protein isselected from a group consisting of a major rib protein and a minor ribprotein, and wherein the major rib protein differentiates from the minorrib protein in that the major rib protein is capable of forming a gasvesicle independently. Preferably, as shown in FIGS. 2 and 3, there isless than 95% homology between the major rib protein and the minor ribprotein, and the Gyp rib protein is derived from P. megaterium.

In exemplary embodiments, the at least two Gyp rib protein can be twomajor rib proteins, two minor rib proteins or a combination of one majorrib protein and one minor rib protein. Preferably, the minor rib proteinforms a complete CGV when the major rib protein is present, and undersuch circumstances the CGV is more stabilized.

In various embodiments, the major rib protein and the minor rib proteinconstitute at least 90% of the chimeric gas vesicle by dry weight.

In one or various embodiments, the minor rib protein comprises a coresequence and a heterologous peptide inserted in frame at C-terminus orN-terminus of the core sequence, wherein the core sequence ischaracterized by a protein structure comprising a first alpha helix, afirst beta sheet at C-terminus of the first alpha helix; a second betasheet at C-terminus of the first beta sheet; and a second alpha helix atC-terminus of the second beta sheet.

In preferred embodiments, the core sequence is selected from a groupconsisting of a GvpA core sequence and a GvpB core sequence.

In various embodiments, wherein the first alpha helix comprises an aminoacid sequence of SSSLAEVIDRILD or SSGLAEVLDRVLD, the first beta sheetcomprises an amino acid sequence of KGIVIDAFARVSL, KGIVIDAFARVSV,KGIVIDAWVRVSL, KGVVVDVWARISL or KGVVVDVWARVSL, the second beta sheetcomprises an amino acid sequence of VGIEILTIEARVVI, VGIELLAIEARIVI orVGIEILTVEARVVA, and the second alpha helix comprises an amino acidsequence of ASVDTWLRYAEXZ, ASVETYLKYAEXZ or ASVDTFLHYAEXZ. Preferably,the X residue is Ala or Glu, and the Z residue is Val or Ile.

To be specific, the core sequence is a hydrophobic region of GvpAprotein or GvpB protein. As illustrated in FIG. 4a , the core sequenceis a tertiary protein structure consisting of 4 secondary proteinstructures arranged in a sequential manner: α-helix, β-sheet, β-sheet,α-helix. The GvpA protein or the GvpB protein is derived fromcyanobacteria, halobacteria or soil bacteria. Preferably, the soilbacteria is Priestia Megaterium; the halobacteria is Halobacteriumsalinarum; the cyanobacteria is Anabaena flos-aquae.

In preferred embodiments, the GvpA protein has an amino acid sequence ofSEQ ID NO.: 1, and the GvpB protein has an amino acid sequence of SEQ IDNO.: 2.

More specifically, the core sequence comprises 9 ^(th) amino acid(Serine) to the 61^(st) amino acid (Valine) of a GvpA protein or a GvpBprotein from P. megaterium but not limited by this. In addition, all gasvesicle rib proteins carry the core sequence. Moreover, any homologs ofGvpA protein or GvpB protein from cyanobacteria, halobacteria or soilbacteria, or any protein sequence homologous to the core sequence, canbe genetically modified and play the role as the minor rib protein.

It should be noted that the secondary structures as mentioned above arepredicted by Jpred 4, an online protein secondary structure predictionprogram. In FIG. 4a , regions forming a-helix are underlined andlabelled as H, while regions forming β-sheet are labelled as S.

Illustrated in FIG. 4b is alignment of the minor rib protein withheterologous peptide, implying a schematic design of the minor ribprotein, wherein the heterologous peptide is inserted at C-terminus ofthe core sequence.

In other embodiments, the heterologous peptide is at least 6 amino acidlong, and the heterologous peptide is derived from a human designedpeptide sequence, a ligand, a hormone, a cytokine, a receptor, aparatope of antibody, a toxic protein, or a fluorescent protein.

In preferred embodiments, the major rib protein is a GvpB protein, andthe minor rib protein is a core sequence with a heterologous peptide,wherein the heterologous peptide has at least 6 amino acids and isinserted in frame at C-terminus or at N-terminus of the core sequence.

Preferably, the core sequence comprises a truncated rib protein keepingthe 1st amino acid (Methionine) to the 61″ amino acid (Valine) of GvpAprotein or GvpB protein from P. megaterium.

Exemplarily, the heterologous peptide of 6 to 56 amino acids or longercan be inserted at C-terminus of the core sequence, and the coresequence is a truncated form of the Gyp rib protein. The truncated Gyprib protein and the heterologous peptide together forms the minor ribprotein which carries a foreign peptide and results in a variety ofapplications in the biotech field. However, longer heterologous peptideleads to less stability of CGV and reduces the yield of CGV production.

In some embodiments, the heterologous peptide can be derived from two ormore heterologous proteins as well. For example, 20-AA from protein Aand 25-AA from protein B form a 45-AA heterologous peptide for theinsertion to form the minor rib protein. Preferably, the heterologouspeptides are derived from the protein sequence of pathogens, variableregions of antibodies, hormones, cytokines and ligands.

In various embodiments, the CGV is expressed in and purified from E.coli.

In one or various embodiments, the chimeric gas vesicle furthercomprises an assembly protein, and the assembly protein comprises a GvpPprotein, a GvpQ protein or a combination thereof. The assembly proteinis derived from P. megaterium. These proteins promote or accelerate orstabilize the formation of the CGV. The minor rib proteins are moreevenly distributed on the CGV's surface with the presence of GvpP andGvpQ proteins. CGVs formed with the assistance of GvpP and GvpQ are morestable than CGVs without the assistance of GvpP and GvpQ.

In one particular embodiment, the GvpP protein has an amino acidsequence of SEQ ID NO.: 3, and the GvpQ protein has an amino acidsequence of SEQ ID NO.: 4.

In preferred embodiments, the CGV further comprises at least oneaccessory protein selected from a group consisting of a GvpR, a GvpN, aGvpF, a GvpG, a GvpL, a GvpS, a GvpK, a GvpJ, a GvpT and a GvpU.

By “chimeric gas vesicles (CGVs)” it means the gas vesicles whichconstitute with at least two kinds of Gyp rib proteins with differentprotein sequences. That is in contrast to all the gas vesicles describedin previous publications which involve only one major rib protein, GvpA.

By “rib protein” it means the gas vesicle protein which forms the “rib”of GV and CGV. Once a gas vesicle forms, the rib protein can't beremoved from a gas vesicle without the destruction of the gas vesicle.The gas vesicle rib protein(s) form ˜4.6-nm ‘ribs’ that stack in arrayand run nearly perpendicular to the long axis of the gas vesicle andconstitute the majority (97%) of the gas vesicle wall. The wall is 2-nmthick and consists of a single layer of the rib protein (FIG. 1). Allgas vesicle rib protein has a hydrophobic tertiary structure including 4secondary structures: α-helix, β-sheet, β-sheet and α-helix. The proteinsequence of the tertiary structure is defined as “core sequence” (FIG.4a, 4b ).

By “major rib protein” it means the GvpA protein in cyanobacteria,halobacteria, and soil bacteria, and GvpB in P. megaterium, or any Gypproteins homologous to and functionally equivalent to the GvpA protein,which forms the rib of the gas vesicle. The gas vesicle major ribprotein is a rib protein with the “core sequence”. Combining with atrace amount of Gyp accessory structure proteins (GvpF, GvpG, GvpJ,GvpK, GvpL, GvpS), it forms complete gas vesicles. It is sufficient toform gas vesicles without the involvement of other rib proteins. Once GVforms, the major rib protein cannot be removed from GV without GVdestruction. The gas vesicle major rib protein constitutes 20 to 99.9%of total protein in the GV. It has been referred to as the majorstructure protein, the major constituent protein, the rib protein, etc.

By “minor rib protein” it means a rib protein with the “core sequence”.It forms a complete gas vesicle only when another kind of rib protein ispresent. Most of the time the other kind of rib protein is a gas vesiclemajor rib protein, but it could be another gas vesicle minor rib proteinwith a different protein sequence. Once CGV forms, the minor rib proteincannot be removed from CGV without CGV destruction. The gas vesicleminor rib protein constitutes 3 to 80% of total protein in the CGV.

The native rib protein is a gas vesicle rib protein, GvpA or itshomolog, found in nature. The truncated rib protein is a native gasvesicle rib protein with part of its amino acid sequence removed. Therecombinant rib protein is a gas vesicle rib protein, either native ortruncated, inserted in frame with a heterologous peptide at C-terminus,N-terminus or in the middle.

By “core sequence” it means the hydrophobic region of GvpA and itshomologs which is a tertiary structure consisting of four secondarystructures: α-helix, β-sheet, β-sheet, α-helix as indicated in FIGS. 4ato 4b . The “core sequence” spans from the 9^(th) amino acid (Serine) tothe 61^(st) amino acid (Valine) of GvpA/B from P. megaterium. All gasvesicle rib proteins carry the core sequence.

In another aspect, the present invention discloses a protein expressionsystem for expression of the chimeric gas vesicle, wherein the proteinexpression system comprises a first polynucleotide fragment encoding oneof the Gyp rib proteins and a second polynucleotide fragment encodingthe other one of the Gyp rib proteins. The protein expression system canbe a DNA-based protein expression system or an RNA-based proteinexpression system.

In various embodiments, the first polynucleotide fragment encoding themajor rib protein; and a second polynucleotide fragment encoding theminor rib protein, wherein the second polynucleotide fragment comprisesa core polynucleotide encoding a core sequence; and a heterologouspolynucleotide fragment encoding a heterologous peptide.

In preferred embodiments, the core sequence is characterized by aprotein tertiary structure comprising a first alpha helix; a first betasheet at C-terminus of the first alpha helix; a second beta sheet atC-terminus of the first beta sheet; and a second alpha helix atC-terminus of the second beta sheet. Preferably, the core sequence isselected from a group consisting of a GvpA core sequence and a GvpB coresequence.

In particular, the core sequence is a hydrophobic region of GvpA proteinor GvpB protein as illustrated in FIG. 4a , wherein the core sequencecomprises 9th to 61st amino acid of a GvpA protein or a GvpB protein.Preferably, the core sequence is a tertiary protein structure consistingof 4 secondary protein structures arranged in a sequential manner:a-helix, β-sheet, β-sheet, a-helix, wherein the GvpA protein or the GvpBprotein is derived from cyanobacteria, halobacteria or soil bacteria.Preferably, the soil bacteria is P. Megaterium; the halobacteria is H.salinarum; the cyanobacteria is A. flos-aquae.

In one preferred embodiment, the first polynucleotide fragment has anucleotide sequence of SEQ ID NO.: 5 or SEQ ID NO.: 6.

In various embodiments, the heterologous peptide is at least 6 aminoacids long, wherein the heterologous peptide is derived from a nativeprotein sequence selected from a group consisting of a ligand, ahormone, a cytokine, a receptor, a paratope of antibody, a toxic proteinand a fluorescent protein, or a human designed peptide sequence.

In some embodiments, the protein expression system further comprises anassembly polynucleotide fragment encoding an assembly protein, whereinthe assembly protein comprises a GvpP protein, a GvpQ protein or acombination thereof. With presence of the assembly proteins, the minorrib proteins are more evenly distributed on the CGV's surface. While inabsence of GvpP or GvpQ protein, CGVs demonstrate less stability andtend to assemble inappropriately or produce less amount than CGVs withthe assistance of the assembly protein.

In certain embodiments, the assembly polynucleotide fragment is derivedfrom P. megaterium gvpP, gvpQ or a combination thereof. Preferably, theassembly polynucleotide fragment has a nucleotide sequence of SEQ IDNO.: 7,

SEQ ID NO.: 8 or SEQ ID NO.: 9.

In some embodiments, the protein expression system further comprises anaccessory polynucleotide fragment encoding at least one accessoryprotein, wherein the accessory protein is selected from a groupconsisting of a GvpR, a GvpN, a GvpF, a GvpG, a GvpL, a GvpS, a GvpK, aGvpJ, a GvpT and a GvpU. In certain embodiments, the accessorypolynucleotide fragment is derived from P. megaterium gvpR, gvpN, gvpF,gvpG, gvpL, gvpS, gvpK, gvpJ, gvpT, gvpU or a combination thereof.Preferably, the accessory polynucleotide fragment has a nucleotidesequence of SEQ ID NO.: 10.

In some embodiments, the protein expression system can be induced in ahost to express the Gyp rib proteins, wherein the host comprises anarchaeon cell, a prokaryotic cell, or a eukaryotic cell. Preferably, thearchaeon cell is H. salinarum; the prokaryotic cell is E. coli; theeukaryotic cells are a mammalian cell, a yeast cell or an insect cell.More preferably, the host is E. coli.

As illustrated in FIG. 8a , the GVs purified from E. coli transformantwith P. megaterium genes: gvpB (encoding a major rib protein) andgvpRNFGLSKJTU. The GVs are generally small (40×40 nm) with some largesized GV (40×200 nm). In an exemplary embodiment, as shown in FIG. 8b ,the CGVs purified from E. coli transformant with P. megaterium genes:gvpA (encoding a recombinant minor rib protein), gvpPQ (assemblyproteins), gvpB (encoding a major rib protein) and gvpRNFGLSKJTU. TheCGVs are large in size (40×200 nm) in the presence of the two Gyp ribproteins and the assembly proteins. In the example as described above,the minor rib protein is a 77-AA truncated GvpA protein fused with a17-AA heterologous peptide.

In some embodiments, to minimize the influence brought by longerheterologous peptide, numbers of amino acid deletion of the Gyp ribprotein shall be considered. Without interruption of CGVs assembly andformation, a maximal deletion at GvpA C-terminus is 25-AA, while themaximal deletion at

GvpB C-terminus is 27-AA. In an exemplary embodiment, maximal GvpAtruncation and maximal heterologous peptide insertion were determined.As shown in TABLE 1, 25 amino acids can be removed from GvpA of P.megaterium without the destruction of CGV, and at least 56-AAheterologous peptide can be inserted into the truncated GvpA atC-terminus without the destruction of CGV. Besides, it can be inferredfrom TABLE 1 that less deletion of rib protein GvpA or GvpB atC-terminus is applicable when shorter heterologous peptide insertion isrequired.

TABLE 1 CGV produc- C- Heter- Preserved tion terminus ologous GvpA(μg/ml Plasmid deletion peptide C-terminus per Name (AA) (AA)AA sequence culture) pNL39 0 0 WLRYAEAVGLLTD 10 KVEEEGLPGRTEE RGAGLSFpNL114 6 0 WLRYAEAVGLLTD 10 KVEEEGLPGRTEE R pNL115 10 0 WLRYAEAVGLLTD 10KVEEEGLPGR pNL119 13 0 WLRYAEAVGLLTD 10 KVEEEGL pNL118 16 0WLRYAEAVGLLTD 10 KVEE pNL105 18 0 WLRYAEAVGLLTD 10 KV pNL106 19 0WLRYAEAVGLLTD 10 K pNL107 20 0 WLRYAEAVGLLTD 10 pNL131 22 0 WLRYAEAVGLL10 pNL132 24 0 WLRYAEAVG 10 pNL135 25 0 WLRYAEAV 10 pNL133 26 0 WLRYAEAN/A pNL136 27 0 WLRYAE N/A pNL134 28 0 WLRYA N/A pNL151 25 47 WLRYAEAV +6 to 8 47-AA pNL148 25 48 WLRYAEAV + 6 to 8 48-AA pNL149 25 49WLRYAEAV + 6 to 8 49-AA pNL150 25 50 WLRYAEAV + 6 to 8 50-AA pNL162 2553 WLRYAEAV + 1 to 2 53-AA pNL163 25 56 WLRYAEAV + 1 to 2 56-AA

In some other embodiments, the protein expression system can be carriedout by two or more different expression vectors for subsequent CGVsexpression in a host. In one exemplary embodiment, a polynucleotidefragment encoding GvpA, GvpP and GvpQ is inserted in a first vector, andanother polynucleotide fragment encoding GvpB, GvpR, GvpN, GvpF, GvpG,GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU is inserted in a second vector.The first vector and the second vector are co-transformed into a hostfor production of the CGVs. Preferably, the vectors are derived fromplasmid pST39 and plasmid pLysS for E. coli host.

By “vector” it means a nucleic acid molecule comprising gene-expressingelements and elements for genetic engineering so as to carry genes ofinterest into a selected host for specific biological functions such asprotein expression or amplifying the copy number of genes of interest.

In other preferred embodiments, as illustrated in FIG. 5, to establish agyp operon system comprising a single operon for protein expression ofthe CGVs, 3′ end of the gvpAPQ polynucleotide fragment or the gypBPQpolynucleotide fragment is covalently linked to 5′ end of thegypBRNFGLSKJTU polynucleotide fragment so that a gvpAPQBRNFGLSKJTUpolynucleotide fragment or a gypBPQBRNFGLSKJTU polynucleotide fragmentis formed.

As illustrated in FIG. 6, to establish the gyp operon system comprisingtwo operons for protein expression of the CGVs when the major ribprotein and the minor rib protein are expressed separately, 3′ end ofthe second polynucleotide fragment is covalently linked to 5′ end of theassembly polynucleotide fragment so as to form a gvpAPQ polynucleotidefragment or a gvpBPQ polynucleotide fragment, while 3′ end of the firstpolynucleotide fragment is covalently linked to 5′ end of the accessorypolynucleotide fragment so as to form a gvpARNFGLSKJTU polynucleotidefragment or a gvpBRNFGLSKJTU polynucleotide fragment.

Practically, an operator or a promoter is required upstream the gvpAPQpolynucleotide fragment, the gvpBPQ polynucleotide fragment, thegvpBRNFGLSKJTU polynucleotide fragment, the gvpAPQBRNFGLSKJTUpolynucleotide fragment and the gypBPQBRNFGLSKJTU polynucleotidefragment so that the gyp operon system can be operated for synthesis ofproteins required for CGV assembly upon induction ofisopropylthiogalactoside (IPTG), lactose, methyl-β-D-thiogalactoside,phenyl-β-D-galactose or ortho-nitrophenyl-β-galactoside (ONPG), but notlimited by this.

In one preferred embodiment, the gvpAPQBRNFGLSKJTU polynucleotidefragment has a 7053-bp DNA sequence encoding GvpA, GvpP, GvpQ, GvpB,GvpR, GvpN, GvpF, GvpG, GvpL, GvpS, GvpK, GvpJ, GvpT and GvpU proteinsand can be inserted into an appropriate expression vector for CGVsprotein expression; preferably the gvpAPQBRNFGLSKJTU polynucleotidefragment has a nucleotide sequence of SEQ ID NO.: 11.

In one another aspect, the present invention as shown in FIG. 9discloses a method for producing the chimeric gas vesicles (CGVs) with aheterologous peptide fused with a genetically engineered gas vesicleminor rib protein GvpA or GvpB, the method comprises steps of:

(Step S1) Construction of plasmid: a plasmid is constructed by insertinga DNA sequence encoding a heterologous peptide having 6 to 56 aminoacids or above in-frame at the C-terminus and/or N-terminus of thegenetically engineered GvpA or GvpB protein in a recombinant gyp operonderived from P. megaterium by PCR and Gibson Assembly.

(Step S2) Transformation of plasmid: the constructed plasmid istransformed into E. coli or other suitable bacteria hosts capable ofexpression and production of CGV.

(Step S3) Induction: the transformant is grown in a proper environmentto an optimal condition for inducing protein expression. Thetransformant is stimulated with IPTG (Isopropylβ-d-1-thiogalactopyranoside) or other inducers for the expression of theCGV.

(Step S4) Purification and quantification of the CGV.

To verify the sequence of the recombinant operon, the method furthercomprises a step of:

(Step S1.1) Sequence validation: The plasmid sequence with desiredinsertion is confirmed by DNA sequencing after step S1.

In some embodiments, the step S1 comprises constructing a CGV expressionplasmid by inserting a DNA sequence encoding the minor rib protein intoa gyp operon vector by PCR and Gibson Assembly, wherein the minor ribprotein has a heterologous peptide at least 6 amino acids (AAs) insertedin frame as a recombinant.

In yet one another aspect, the present invention as shown in FIG. 10discloses a method for eliciting immune response by administratingCGV-Covid-19 to a subject in need, the method comprising steps of:

(Step A) Purification and quantification of CGV-Covid-19 vaccine in PBS.

(Step B) Administration of the CGV-Covid-19 vaccine into the subject inneed.

(Step C) Detection of the immune responses on the subject.

In a certain embodiment, CGV-Covid-19 is administrated on the subject inneed via intranasal administration, wherein a 17-AA heterologous peptidederived from S-protein of Covid-19 virus is inserted at C-terminus ofthe core sequence so that the CGV presents an epitope derived fromSARS-CoV-2 (Covid-19) spike protein. Enzyme-linked immunosorbent assay(ELISA) is applied to measure elicitation of the immune responses by theCGV-Covid-19 vaccine, but the measure for detection is not limited bythis. The subject in need includes human, mice, canine, porcine,chimpanzee or other mammal.

The CGVs of the invention may be used as vaccines for infectiousdiseases, therapeutic vaccines for cancer and other diseases. They canbe therapeutic agents (such as carrying hormones, cytokines, ligands, orpart of variable regions of antibodies) for cancer, metabolic diseases,and other diseases. They can also function as non-invasive contrastagents for ultrasound, MRI and other imaging technologies. In short, awide variety of applications are possible with the CGVs used as ananoparticle.

Example 1

Construction and Confirmation of Plasmid

Any effective E. coli plasmid/host expression system can be used, suchas pBB322 derived plasmids/BL21 E. coli strain (New England Biolab),pET28 plasmid/BL21(A1) E. coli strain (Thermo Fisher) or pST39plasmid/Rosetta E. coli strain (Millipore Sigma).

The plasmid was digested with restriction enzyme/enzymes or PCR withparticular primers to generate the corresponding DNA fragment. Theproper DNA primers encoding a peptide of interest were designed andordered from any commercial primer vendors by one of ordinary skill inthe art.

The DNA fragment encoding heterologous truncated GvpA protein or GvpBprotein with a peptide of interest, and a DNA fragment carrying the restof 13 gyp genes: gvpPQBRNFGLSKJTU from P. megaterium were amplified byPCR with the proper DNA primers. The fragments were subsequently ligatedby Gibson Assembly (Thermo Fisher). The Gibson Assembly mixture wastransformed into proper E. coli competent cells and transformants wereanalyzed and sequenced by one of ordinary skill in the art.

Expression and Production of CGV

The plasmids with the correct DNA sequence insertion were transformedinto desired E. coli hosts. A single colony was picked and inoculatedwith LB media with appropriate selection antibiotics. CGV expression canbe induced by IPTG or other inducers and purification is done by amodified protocol. The protocol including following steps:

a. Transformed E. coli were cultured for 20 to 22 hours and thencentrifuged at 500 g for 1 to 2 hours.

b. The pellet was resuspended in PBS buffer and lysed by detergent,lysozyme and DNase.

c. The cell lysate was centrifuged at 500 g for 5 to 30 minutes so thatCGV was isolated from cell debris. Of note, the CGV floated at the topof the lysate suspension.

d. The cell debris and the midnatant were removed by a needle.

e. The remaining CGV was suspended in PBS.

f. Step c to Step e were repeated for 10 times and 99% purity of CGV wasobtained.

g. Final concentration of CGV was measured by a spectrophotometer atOD₅₀₀(NanoDrop ND-1000, Thermo Scientific). Of note, one OD₅₀₀ of CGV isequivalent to 145 μg/ml of protein.

Example 2

To verify that insertion of heterologous peptide from one foreignprotein or more foreign proteins can be realized in the presentinvention, in example 2, one 20-AA peptide from H1N1 virus HA(hemagglutinin) protein and another 20-AA peptide from Covid-19 virusSpike protein were linked to form a 40-AA insertion peptide. Then, theminor rib genes with heterologous peptide from two foreign proteins wereinserted into the 14 gene gyp operon on the same plasmid or on twoindividual plasmid vectors. Transformation or co-transformation of theplasmid(s) into E. coli competent cells such that CGV particles carryingtwo or more heterologous peptides were brought on mass production.

Example 3

To further validate protein expression of CGVs carrying variousheterologous peptides, CGVs produced and isolated by procedures asdescribed in example 1 were confirmed by Western Dot Blot.

TABLE 2 Heter- ologous CGV Peptide S-Protein Dot samples SequencesSequence 1 Positive Covid-19 S-Protein control S-protein 2 pNL39 GvpA Nowithout hetero insertion geneous of peptide hetero- sequence geneoussequence 3 pNL66 TGCVIAW T430 to NSNNLDS S443 4 pNL67 NLDSKVG N440 toGNYNYLY Y453 5 pNL68 NYLYRLF N450 to RKSNLKP P463 6 pNL69 NLKPFERN460 to DISTEIY Y473 7 pNL70 TEIYQAG T470 to STPCNGV V483 8 pNL71CNGVEGF C480 to NCYFPLQ Q493 9 pNL72 FPLQSYG F490 to FQPTNGV V50310 pNL73 TNGVGYQ T500 to PYRVVVL S513

CGV samples with various heterologous peptides were purified from the E.coli culture transformed with the corresponding plasmid constructs. TheWestern Dot Blot was done with rabbit anti-Covid-19 S protein as theprimary antibody and goat anti-rabbit IgG-HRP as secondary antibody.

As shown in FIG. 7, dots representing positive CGVs were Dot 5 to 8,which suggests that antigen region of Covid-19 S-protein is between450^(th) to 493^(rd) AA. In TABLE 2, detailed information of Covid-19 Sprotein sequences serving as inserts in recombinant gyp operons werelisted down, and antigen regions of Covid-19 S protein were identifiedas NYLYRLFRKSNLKP (N450 to P463), NLKPFERDISTEIY (N460 to Y473),TEIYQAGSTPCNGV (T470 to V483) and CNGVEGFNCYFPLQ (C480 to Q493),respectively.

The advantages of the present invention, chimeric gas vesicles (CGVs),over traditional GV and GVNP are numerous.

For example, previous attempts of direct genetic modification of GvpA inthe conventional GV are unsuccessful due to the rigidity of GvpA in theGV. To date, no more than 5-AA heterologous peptide can be inserted atthe C-terminus of GvpA without the destruction of GV. The CGV disclosedin the present invention overcomes this stability limitation and makesthe CGVs possible for various applications. In GV research inhalobacteria, the genetically modified GvpC of GVNP attaches to GV viaprotein-protein interaction, and is vulnerable to thermohalinefluctuations. This makes the GvpC easy to fall off from the GVNP, thusgreatly reduces the possibility of using the conventional GVs forapplications. In the CGVs, the heterologous peptide can be inserted orfused covalently with the truncated/native GvpA or its homolog to formthe rib protein. As a part of the rib structure, the fall-off problem ofthe heterologous peptide present in the conventional GVs is eliminated.For industrial or commercial productions, the making of the CGVs can beaccomplished in much shorter production cycle. In some instances, it canbe produced within 2 days, which is drastically shorter than theproduction cycle of GVNPs in halobacteria or GVs in cyanobacteria. TheCGVs are more stable than the conventional GVs. The CGVs produced in E.coli with gyp operon from P. megaterium are more stable than any otherGVs previously known. The CGVs of the present invention have a higher“critical collapse pressure” than the conventional GVs. For storage, theCGVs in one of the embodiments maintain their integrity at roomtemperature for at least one month, and at 4° C. for at least sixmonths, without any degradation. All these features of the CGVs of thepresent invention increases immensely the possibilities of itsapplications in the future.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

What is claimed is:
 1. A chimeric gas vesicle (CGV) comprising at leasttwo Gyp rib proteins, wherein the rib protein is selected from a groupconsisting of a major rib protein and a minor rib protein, and whereinthe major rib protein differentiates from the minor rib protein in thatthe major rib protein is capable of forming a gas vesicle independently.2. The chimeric gas vesicle as claimed in claim 1, wherein any one ofthe Gyp rib proteins is derived from P. megaterium.
 3. The chimeric gasvesicle as claimed in claim 2, wherein the minor rib protein comprises acore sequence and a heterologous peptide inserted in frame at C-terminusor N-terminus of the core sequence.
 4. The chimeric gas vesicle asclaimed in claim 3, wherein the core sequence comprises a GvpA coresequence.
 5. The chimeric gas vesicle as claimed in claim 3, wherein thecore sequence comprises 9^(th) to 61^(st) amino acid of a GvpA proteinderived from P. megaterium.
 6. The chimeric gas vesicle as claimed inclaim 3, wherein the heterologous peptide is at least 6 amino acidslong.
 7. The chimeric gas vesicle as claimed in claim 4, wherein theheterologous peptide is derived from a native peptide sequence selectedfrom a group consisting of, a ligand, a hormone, a cytokine, a receptor,a paratope of antibody, a toxic protein and a fluorescent protein, or ahuman designed peptide sequence.
 8. The chimeric gas vesicle as claimedin claim 1, wherein the CGV is expressed in and purified from E. coli.9. The chimeric gas vesicle as claimed in claim 1, wherein the CGV isassembled with assistance of at least one assembly protein.
 10. Thechimeric gas vesicle as claimed in claim 9, wherein the assembly proteincomprises a GvpP protein, a GvpQ protein or a combination thereof. 11.The chimeric gas vesicle as claimed in claim 1, wherein the major ribprotein and the minor rib protein form at least 90% of the chimeric gasvesicle by dry weight.
 12. A protein expression system for expressingthe CGV as claimed in claim 1, comprising: a first polynucleotidefragment encoding one of the Gyp rib proteins; and a secondpolynucleotide fragment encoding the other one of the Gyp rib proteins.13. The protein expression system as claimed in claim 12, wherein thesecond polynucleotide fragment comprises: a core polynucleotide fragmentencoding a core sequence; and a heterologous polynucleotide fragmentencoding a heterologous peptide.
 14. The protein expression system asclaimed in claim 13, wherein the core sequence comprises a GvpA coresequence.
 15. The protein expression system as claimed in claim 13,wherein the core sequence comprises 9^(th) to 61st amino acid of a GvpAprotein derived from P. megaterium.
 16. The protein expression system asclaimed in claim 13, wherein the heterologous peptide is at least 6amino acids long.
 17. The protein expression system as claimed in claim13, wherein the heterologous peptide is derived from a native peptidesequence selected from a group consisting of a ligand, a hormone, acytokine, a receptor, a paratope of antibody, a toxic protein and afluorescent protein, or a human designed peptide sequence.
 18. Theprotein expression system as claimed in claim 13, further comprising anassembly polynucleotide fragment encoding an assembly protein.
 19. Theprotein expression system as claimed in claim 13, wherein the CGV isexpressed in and purified from E. coli.
 20. A chimeric gas vesicle (CGV)expressed in and purified from E. colicomprising at least two Gyp ribproteins, wherein the rib protein is selected from a group consisting ofa major rib protein and a minor rib protein, wherein the major ribprotein differentiates from the minor rib protein in that the major ribprotein is capable of forming a gas vesicle independently, and whereinone of the rib proteins having a core sequence with a heterologouspeptide inserted in frame at C-terminus or N-terminus of the coresequence.